Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

Regulation of the cardiac calcium channel by protein phosphatases

The calcium current (ICa) through the L-type channel in cardiac ventricular cells is enhanced by phosphorylation of a channel protein [Kameyama, M., Hofmann, F. & Trautwein, W. (1985) Pflügers Arch. Eur. J. Physiol. 405, 285-293]. We investigated the possible contribution of the 'catalytic subunits' of protein phosphatase 1 and 2A in the down-regulation of the cardiac calcium channel. Single guinea-pig ventricular myocytes were voltage clamped and the following results were obtained. (1) Intracellular perfusion of the myocyte with the catalytic subunits of protein phosphatase 1 (2 microM) as well as 2A (2.3 microM) completely abolished the increase of ICa induced by isoprenaline (0.05 microM) but did not decrease the basal level of ICa. Alkaline and acid phosphatases were without detectable effect. (2) Cell dialysis with the modulator of protein phosphatase 1 (inhibitor-2) under control conditions (without addition of isoprenaline) caused a slow significant increase of ICa. (3) The time course for the wash-out of the isoprenaline effect was considerably prolonged in the presence of high concentrations of inhibitor-2. (4) Perfusion of the myocyte under basal conditions with adenosine 5'-[gamma-thio]triphosphate led to a slow increase of ICa. Additional superfusion of the cell with a threshold concentration of isoprenaline (0.01 microM) resulted in a rapid increase of ICa which could not be washed out during at least 10 min. From these results we make the following conclusions. (1) The calcium channel from guinea-pig myocytes is regulated by phosphorylation-dephosphorylation. (2) The catalytic subunits of the protein phosphatases 1 as well as 2A, purified from rabbit skeletal muscle, catalyse the down-regulation of the channel. (3) Indirect evidence suggests that endogenous protein phosphatase 1 contributes only partially to the dephosphorylation of the calcium channel in the intact myocyte.     

Targeting of PKA, PKC and protein phosphatases to cellular microdomains

The intracellular responses to many distinct extracellular signals involve the direction of broad-based protein kinases and protein phosphatases to catalyse quite specific protein phosphorylation/dephosphorylation events. It is now clear that such specificity is often achieved through subcellular targeting of distinct pools of kinase or phosphatase towards particular substrates at specific subcellular locations. Given the dynamic nature of protein phosphorylation reactions, coordinated control of both kinase and phosphatases is often required and complexes formed by common scaffold or targeting proteins exist to direct both kinase and phosphatase to the same subcellular location. In many cases more than one kinase or phosphatase is required and binding proteins which target more than one kinase or phosphatase have now been identified. This review summarizes recent findings relating to the concept of targeting PKA, PKC and the major serine/threonine phosphatases, PP1, PP2A and PP2B, through the formation of multi-enzyme signalling complexes.     

Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes

We investigated the ability of pyrroloquinoline quinone (PQQ) to confer resistance to acute oxidative stress in freshly isolated adult male rat cardiomyocytes. Fluorescence microscopy was used to detect generation of reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsi(m)) depolarization induced by hydrogen peroxide. H(2)O(2) caused substantial cell death, which was significantly reduced by preincubation with PQQ. H(2)O(2) also caused an increase in cellular ROS levels as detected by the fluorescent indicators CM-H2XRos and dihydroethidium. ROS levels were significantly reduced by a superoxide dismutase mimetic Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) or by PQQ treatment. Cyclosporine-A, which inhibits mitochondrial permeability transition, prevented H(2)O(2)-induced Deltapsi(m) depolarization, as did PQQ and MnTBAP. Our results provide direct evidence that PQQ reduces oxidative stress, mitochondrial dysfunction, and cell death in isolated adult rat cardiomyocytes. These findings provide new insight into the mechanisms of PQQ action in the heart.     

Parthenolide induces a distinct pattern of oxidative stress in cardiac myocytes

Although parthenolide was reported to reduce cardiovascular damage in endotoxic shock and have beneficial effects in myocardial ischemia, its actions on cardiac myocytes have not been reported. Because parthenolide possesses an alpha-methylene-gamma-lactone ring and epoxide residue, we hypothesized that it would induce oxidative stress in cardiac myocytes. Superoxide production and sources, viability, glutathione levels, and mitochondrial membrane potential were studied in neonatal rat ventricular myocytes treated with parthenolide. Parthenolide, dose dependently, induced oxidase activity as assessed by superoxide generation in cell lysates. Superoxide formation was increased more than 4-fold with 50 microM parthenolide. At concentrations >5 microM, parthenolide decreased cell viability in a dose-and time-dependent manner, and activated the stress MAP kinases JNK and p38. Over 6 h, parthenolide at concentrations >5 microM markedly depleted intracellular glutathione and led to collapse of the mitochondrial membrane potential. At lower parthenolide concentrations (<5 microM) the source of superoxide was mitochondria; at higher concentrations (>5 microM) the primary source was NADPH oxidase. We conclude that parthenolide causes oxidative stress in cardiac myocytes by inducing superoxide formation by mitochondrial and NADPH oxidase in a dose-dependent manner. Parthenolide may be a useful tool for studying the roles of oxidative stress and mitochondrial dysfunction in the pathogenesis of cardiac hypertrophy and failure.     

Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress.

Protein S-glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol-group modifications. Special attention will be paid to recently published work supporting a role for S-glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S-glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post-translational protein modification will be discussed.     

Mechanically induced orientation of adult rat cardiac myocytes in vitro

Summary A population of freshly isolated adult rat cardiac myocytes is spatially oriented using a computerized mechanical cell stimulator device for tissue cultured cells. A continuous unidirectional stretch of the substratum at 60 to 400 μm/min for 120 to 30 min, respectively, during the cell attachment period in serum-free medium induces a significant three-fold increase in the number of rod-shaped myocytes oriented parallel to the direction of movement. The myocytes orient less well with unidirectional substratum stretching after their adhesion to the substratum. In contrast, adult myocytes plated onto a substratum undergoing continuous 10% stretch-relaxation cycling show no significant change in myocyte orientation or cytoskeletal organization. Orientation of rod-shaped myocytes is dependent on several factors other than the type of mechanical activity. These include: a) the speed of substratum movement; b) the final stretch amplitude; and c) the timing between initiation of substratum stretching and adhesion of myocytes to the substratum. Oriented adult rod shaped myocytes representing 65 to 70% of the total myocyte population in this model system can now be submitted to different patterns of repetitive mechanical stimulation for the study of stretch-induced alterations in cell growth and gene expression. This work was supported by grants AR36266, AR39998, and RR05818 from the National Institutes of Health, Bethesda, MD, and grant NAG2-414 from the National Aeronautics and Space Administration, Washington, DC. J.-L. Samuel was a recipient from the Foundation pour la Recherche Médicale.     

Regulation of cAMP homeostasis by the efflux protein MRP4 in cardiac myocytes

Recent studies indicate that members of the multidrug-resistance protein (MRP) family belonging to ATP binding cassette type C (ABCC) membrane proteins extrude cyclic nucleotides from various cell types. This study aimed to determine whether MRP proteins regulate cardiac cAMP homeostasis. Here, we demonstrate that MRP4 is the predominant isoform present at the plasma membrane of cardiacmyocytes and that it mediates the efflux of cAMP in these cells. MRP4-deficient mice displayed enhanced cardiac myocyte cAMP formation, contractility, and cardiac hypertrophy at 9 mo of age, an effect that was compensated transiently by increased phosphodiesterase expression at young age. These findings suggest that cAMP extrusion via MRP4 acts together with phosphodiesterases to control cAMP levels in cardiac myocytes.     

5'-AMP-activated protein kinase is inactivated by adrenergic signalling in adult cardiac myocytes

In adult rat cardiac myocytes adrenaline decreased AMPK (AMP-activated protein kinase) activity with a half-time of approximately 4?min, decreased phosphorylation of AMPK (α-Thr172) and decreased phosphorylation of ACC (acetyl-CoA carboxylase). Inactivation of AMPK by adrenaline was through both α1- and β-ARs (adrenergic receptors), but did not involve cAMP or calcium signalling, was not blocked by the PKC (protein kinase C) inhibitor BIM I (bisindoylmaleimide I), by the ERK (extracellular-signal-regulated kinase) cascade inhibitor U0126 or by PTX (pertussis toxin). Adrenaline caused no measurable change in LKB1 activity. Adrenaline decreased AMPK activity through a process that was distinct from AMPK inactivation in response to insulin or PMA. Neither adrenaline nor PMA altered the myocyte AMP:ATP ratio although the adrenaline effect was attenuated by oligomycin and by AICAR (5-amino-4-imidazolecarboxamide-1-β-D-ribofuranoside), agents that mimic 'metabolic stress'. Inactivation of AMPK by adrenaline was abolished by 1?μM okadaic acid suggesting that activation of PP2A (phosphoprotein phosphatase 2A) might mediate the adrenaline effect. However, no change in PP2A activity was detected in myocyte extracts. Adrenaline increased phosphorylation of the AMPK β-subunit in vitro but there was no detectable change in vivo in phosphorylation of previously identified AMPK sites (β-Ser24, β-Ser108 or β-Ser182) suggesting that another site(s) is targeted.     

Importance of endogenous substrates for cultured adult rat cardiac myocytes

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM medium concentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.     

Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress

Localization and staining features of the oxidant-sensitive fluorescent probe 2'7'-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.     

Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial K_ATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

     

Necrotic death without mitochondrial dysfunction-delayed death of cardiac myocytes following oxidative stress

Oxidative stress has been implicated in cell death in range of disease states including ischemia/reperfusion injury of the heart and heart failure. Here we have investigated the mechanisms of cell death following chronic exposure of cardiac myocytes to oxidative stress initiated by hydrogen peroxide. This exposure induced a delayed form of cell death with ultrastructural changes typical of necrosis, and that was accompanied by the release of lactate dehydrogenase and increased lipid peroxidation. However, this delayed death was not accompanied by the loss of mitochondrial membrane potential or caspase-3 activation. Furthermore, we could demonstrate that this delayed necrosis was at least partially prevented by pre-treatment with the hypertrophic stimuli endothelin-1 or leukemic inhibitory factor. Our results suggest that this delayed form necrosis may also comprise an ordered series of events involving pathways amenable to therapeutic modulation.     

Osmotic stress induces loss of glutathione and increases the sensitivity to oxidative stress in H9c2 cardiac myocytes

It has been observed that H9c2 cardiac cells cultured in physiologic solutions exhibit delayed cell death after repeated medium replacements, of which the cause was the relatively mild osmotic challenges during the renewal of the culture medium. Interestingly, the cell damage was associated with altered intracellular GSH homeostasis. Therefore, this study attempted to elucidate the effects of osmotic stress on GSH metabolism. In cells subjected to osmotic stress by lowering the NaCl concentration of the medium, the cell swelling was rapidly counterbalanced, but the intracellular GSH content was significantly lower in 3 h. Meanwhile, the ratio of GSH-to-GSSG was not affected. As expected, osmotic stress also increased the sensitivity to H₂O₂, which was attributable to the decrease of GSH content. The decrease of GSH content was similarly evident when the synthetic pathways of GSH were blocked by BSO or acivicin. It was concluded that osmotic stress induced the decrease of intracellular GSH content by increased consumption and this loss of GSH rendered the cells susceptible to a subsequent oxidative stress.     

Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes

Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply prime PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.     

Regulation of protein tyrosine phosphatases by reversible oxidation

Oxidation of the catalytic cysteine of protein-tyrosine phosphatases (PTP), which leads to their reversible inactivation, has emerged as an important regulatory mechanism linking cellular tyrosine phosphorylation and signalling by reactive-oxygen or -nitrogen species (ROS, RNS). This review focuses on recent findings about the involved pathways, enzymes and biochemical mechanisms. Both the general cellular redox state and extracellular ligand-stimulated ROS production can cause PTP oxidation. Members of the PTP family differ in their intrinsic susceptibility to oxidation, and different types of oxidative modification of the PTP catalytic cysteine can occur. The role of PTP oxidation for physiological signalling processes as well as in different pathologies is described on the basis of well-investigated examples. Criteria to establish the causal involvement of PTP oxidation in a given process are proposed. A better understanding of mechanisms leading to selective PTP oxidation in a cellular context, and finding ways to pharmacologically modulate these pathways are important topics for future research.     

Oxidation of myoglobin in isolated adult rat cardiac myocytes by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid

The oxidation of intracellular myoglobin by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was studied in suspensions of isolated adult rat heart cells. Myoglobin was converted to a species identified as ferrylMb by its reaction with Na2S to form ferrous sulfmyoglobin. This process was time-dependent and concentration-dependent in a manner consistent with direct accessibility of the exogenous peroxide to the cytosolic protein. The results indicate that myoglobin oxidation may be an early sign of oxidative injury and may limit myocardial function by elimination of this short-term O2 reserve.